**3. Natural ligands of tetraspanin proteins**

#### **3.1 The tetraspanin web and intertetraspanin contacts**

The ability of members of the tetraspanin family to assemble into a unique biological feature known as tetraspanin-enriched microdomain (TEM) is due to their mutual interactions; however, these structures include also receptors, integrins and signaling molecules such as phosphatidyl-kinase C (PKC) and phosphatidylinositol-4-kinase (PI4K) [9]. These interactions are fundamental for cellular functions such as cell adhesion, proliferation and motility. Interactions between tetraspanin members are important in maintaining the integrity and stability of the tetraspanin web and providing binding sites for different ligands. The multimers of newly synthesized proteins are formed in the Golgi apparatus. The predominantly cross-linked tetraspanin species are homodimers, but also higher order complexes and low amounts of heterodimeric tetraspanins (CD81/ CD9, CD9/CD151, CD81/CD151) were identified [27]. It has been suggested that tetraspanin homodimers, formed in the Golgi and present at the cell surface, serve as building blocks in the assembly of higher organized tetraspanin protein complexes. Interestingly, the exosomes originating from cell lines overexpressing CD9 are believed to be enriched in more stable TEMs [10]. Overall, although most tetraspanins can interact with most other tetraspanins, and similarly engage with several other proteins, the nature of these interactions has been until recently classified only according to their stability in the presence of detergents of different stringency, which does not necessarily reflect their significance in the cellular milieu [34]. A thorough characterization of strength and abundance of the interactions between the members participating in a tetraspanin web in a particular cell and physiological situation is therefore needed and will support the understanding of its mediated biological effects. Similarly, most data on tetraspanin functionality come from studies on their localization on cell membranes, while functional data in vesicles are still scarce. Therefore, we here summarize the known cellular functions, while speculating how this might translate to EVs.

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*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting*

lar dynamic simulation of the cell membrane-embedded protein [37].

**role of tetraspanins in cancer and metastasis**

**3.2 Interaction of tetraspanins with integrins and matrix-degrading enzymes:** 

Important association partners of tetraspanins are the integrins. The role of such complexes in invasive growth *in vivo* as well as the effect of integrin-mediated binding events on cell proliferation and invasion is well established. Especially, the laminin-binding integrins (α6β4, α3β1, α6β1 and α7β1) exhibit extensive interactions with tetraspanin proteins [12, 38]. The functionality of integrins may depend critically on their interaction of tetraspanins: it has early been described that the remarkably stable association of the tetraspanin CD151 and the integrin α3β1 leads to a high level of activation of cellular PI4K [39]. Further, CD151 interacts directly with the α<sup>3</sup> subunit and links it to other tetraspanins, CD9 and CD81. Loss of CD151 abrogates the α3β1 mediated mobility on its ligands, laminin-332 and laminin-551. CD9/CD81 complex may even regulate the integrin-mediated functions independently of CD151 by forming a complex with the integrin and directing the PKCα-α3β1 association [40]. Another example of tetraspanin-integrin association reveals its proangiogenic role through VEGF induction, mediated by cooperation between TM4SF5 and integrin α5 of epithelial cells [41]. Interestingly, removal of CD151 palmitoylation sites did not disrupt the CD151–α6β4 complex in epithelial cells but strongly influenced α6β4 integrin–dependent cell morphology [42]. The rat tetraspanin D6.1A (human homolog is CO-029) was able to induce systemic angiogenesis by initiation of an angiogenic loop that reached organs distant from the tumor, probably due to the abundance of D6.1A in tumor-derived exosomes. This is in line with reports claiming that EVs prepare niches for metastatic tumor cells at tissues distant from the primary tumor [43]. This tetraspanin associates with integrins α3β1, α6β1 and α6β4, as well as with tetraspanins CD9 and CD81, and is similarly to CD151 linked to tumor-promoting activities [44]. Active complexes of tetraspanins and integrins influence biological processes other than cellular signaling by interacting with cellular metalloproteinases, important players in the remodeling of extracellular matrix. A study of MDA-MB-231 cells, a breast cancer cell line, has indicated that the α3β1-tetraspanin protein complex may be linked to an invasive phenotype of tumor cells via modulation of various signaling pathways, including activation of membrane metalloproteinase-2 (MMP-2), an enzyme associated with invasive migration of the cells, and affecting phosphatidylinositol-3-kinase (PI3K) signaling pathways, which control actin cytoskeleton dynamics [45]. By the incorporation of the members of a disintegrin and metalloproteinase (ADAM) family members the tetraspanins are able to influence the

An important step towards the understanding of the specificity of the tetraspanin interactions in TEM has been achieved by delineation of the involved tetraspanin regions by dissecting the model tetraspanin into domains, differently amenable for modification. Early experiments that addressed the relatively unstructured and at the same time antigen-binding competent regions that appeared attractive for mutagenesis resulted in a protein that showed aberrant clustering involving both homo- and heterodimerization of resulting full-length tetraspanins [35], albeit the mutagenesis method employed in this study was a complete deletion of targeted domains. The CD81 D-region was studied in more detail: the CD9 and CD151 tetraspanins were more competent of clustering with CD81 when homologously engrafted with CD81 D-region [36]. When the mutagenized CD81 EC2 molecular subunits were transplanted to other tetraspanins, the extremely flexible conformation of the solvent-exposed D-segment of CD81 EC2 was sufficient to overcome the orientational restrictions to initiate the homotypic contact for dimerization, and this finding has been corroborated with both wet-lab data and the insights from molecu-

*DOI: http://dx.doi.org/10.5772/intechopen.83537*

#### *Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting DOI: http://dx.doi.org/10.5772/intechopen.83537*

An important step towards the understanding of the specificity of the tetraspanin interactions in TEM has been achieved by delineation of the involved tetraspanin regions by dissecting the model tetraspanin into domains, differently amenable for modification. Early experiments that addressed the relatively unstructured and at the same time antigen-binding competent regions that appeared attractive for mutagenesis resulted in a protein that showed aberrant clustering involving both homo- and heterodimerization of resulting full-length tetraspanins [35], albeit the mutagenesis method employed in this study was a complete deletion of targeted domains. The CD81 D-region was studied in more detail: the CD9 and CD151 tetraspanins were more competent of clustering with CD81 when homologously engrafted with CD81 D-region [36]. When the mutagenized CD81 EC2 molecular subunits were transplanted to other tetraspanins, the extremely flexible conformation of the solvent-exposed D-segment of CD81 EC2 was sufficient to overcome the orientational restrictions to initiate the homotypic contact for dimerization, and this finding has been corroborated with both wet-lab data and the insights from molecular dynamic simulation of the cell membrane-embedded protein [37].
